Peptide antagonists of zonulin and methods for use of the same

ABSTRACT

Peptide antagonists of zonulin are disclosed, as well as methods for the use of the same. The peptide antagonists bind to the  zonula occludens  receptor, yet do not physiologically modulate the opening of mammalian tight junctions.

The development of the present invention was supported by the Universityof Maryland, Baltimore, Md. The invention described herein was supportedby funding from the National Institutes of Health (NIH DK-48373). TheGovernment has certain rights.

FIELD OF THE INVENTION

The present invention relates to peptide antagonists of zonulin, as wellas methods for the use of the same. Said peptide antagonists bind to thezonula occludens receptor, yet do not physiologically modulate theopening of mammalian tight junctions.

BACKGROUND OF THE INVENTION I. Function and Regulation of IntestinalTight Junctions

The tight junctions (“tj”) or zonula occludens (hereinafter “ZO”) areone of the hallmarks of absorptive and secretory epithelia (Madara, J.Clin. Invest., 83:1089-1094 (1989); and Madara, Textbook of SecretoryDiarrhea Eds. Lebenthal et al, Chapter 11, pages 125-138 (1990). As abarrier between apical and basolateral compartments, they selectivelyregulate the passive diffusion of ions and water-soluble solutes throughthe paracellular pathway (Gumbiner, Am. J. Physiol., 253 (Cell Physiol.22):C749-C758 (1987)). This barrier maintains any gradient generated bythe activity of pathways associated with the transcellular route(Diamond, Physiologist, 29:10-18 (1977)).

Variations in transepithelial conductance can usually be attributed tochanges in the permeability of the paracellular pathway, since theresistances of enterocyte plasma membranes are relatively high (Nadara,supra). The ZO represents the major barrier in this paracellularpathway, and the electrical resistance of epithelial tissues seems todepend on the number of transmembrane protein strands, and theircomplexity in the ZO, as observed by freeze-fracture electron microscopy(Madara at al, J. Cell Biol., 101:2124-2133 (1985)).

There is abundant evidence that ZO, once regarded as static structures,are in fact dynamic and readily adapt to a variety of developmental(Magnuson et al, Dev. Biol., 67:214-224 (1978); Revel et al, Cold SpringHarbor Symp. Quant. Biol., 40:443-455 (1976); and Schneeberger et al, J.Cell Sci., 32:307-324 (1978)), physiological (Gilula et al, Dev. Biol.,50:142-168 (1976); Madara et al, J. Membr. Biol., 100:149-164 (1987);Mazariegos et al, J. Cell Biol., 98:1865-1877 (1984); and Sardet et al,J. Cell Biol., 80:96-117 (1979)), and pathological (Milks et al, J. CellBiol., 103:2729-2738 (1986); Nash at al, Lab. Invest., 59:531-537(1988); and Shasby at al, Am. J. Physiol., 255 (Cell Physiol.,24):C781-C788 (1988)) circumstances. The regulatory mechanisms thatunderlie this adaptation are still not completely understood. However,it is clear that, in the presence of Ca²⁺, assembly of the ZO is theresult of cellular interactions that trigger a complex cascade ofbiochemical events that ultimately lead to the formation and modulationof an organized network of ZO elements, the composition of which hasbeen only partially characterized (Diamond, Physiologist, 20:10-18(1977)). A candidate for the transmembrane protein strands, occludin,has recently been identified (Furuse et al, J. Membr. Biol., 87:141-150(1985)).

Six proteins have been identified in a cytoplasmic submembranous plaqueunderlying membrane contacts, but their function remains to beestablished (Diamond, supra). ZO-1 and ZO-2 exist as a heterodimer(Gubiner et al, Proc. Natl. Acad. Sci., USA, 88:3460-3464 (1991)) in adetergent-stable complex with an uncharacterized 130 kD protein (ZO-3).Most immunoelectron microscopic studies have localized ZO-1 to preciselybeneath membrane contacts (Stevenson et al, Molec. Cell Biochem.,83:129-145 (1988)). Two other proteins, cingulin (Citi at al, Nature(London), 333:272-275 (1988)) and the 7H6 antigen (Zhong et al, J. CellBiol., 120:477-483 (1993)) are localized further from the membrane andhave not yet been cloned. Rab 13, a small GTP binding protein has alsorecently been localized to the junction region (Zahraoui et al, J. CellBiol., 124:101-115 (1994)). Other small GTP-binding proteins are knownto regulate the cortical cytoskeleton, i.e., rho regulatesactin-membrane attachment in focal contacts (Ridley et al, Cell,70:389-399 (1992)), and rac regulates growth factor-induced membraneruffling (Ridley et al, Cell, 70:401-410 (1992)). Based on the analogywith the known functions of plaque proteins in the better characterizedcell junctions, focal contacts (Guan et al, Nature, 358:690-692 (1992)),and adherens junctions (Tsukita et al, J. Cell Biol., 121:1049-1053(1993)), it has been hypothesize that tj-associated plaque proteins areinvolved in transducing signals in both directions across the cellmembrane, and in regulating links to the cortical actin cytoskeleton.

To meet the many diverse physiological and pathological challenges towhich epithelia are subjected, the ZO must be capable of rapid andcoordinated responses that require the presence of a complex regulatorysystem. The precise characterization of the mechanisms involved in theassembly and regulation of the ZO is an area of current activeinvestigation.

There is now a body of evidence that tj structural and functionallinkages exist between the actin cytoskeleton and the tj complex ofabsorptive cells (Gumbiner at al, supra; Madara et al, supra; andDrenchahn et al, J. Cell Biol., 107:1037-1048 (1988)). The actincytoskeleton is composed of a complicated meshwork of microfilamentswhose precise geometry is regulated by a large cadre of actin-bindingproteins. An example of how the state of phosphorylation of anactin-binding protein might regulate cytoskeletal linking to the cellplasma membrane is the myristoylated alanine-rich C kinase substrate(hereinafter “MARCKS”). MARCKS is a specific protein kinase C(hereinafter “PKC”) substrate that is associated with the cytoplasmicface of the plasma membrane (Aderem, Elsevier Sci. Pub. (UK), pages438-443 (1992)). In its non-phosphorylated form, MARCKS crosslinks tothe membrane actin. Thus, it is likely that the actin meshworkassociated with the membrane via MARCKS is relatively rigid (Hartwig etal, Nature, 356:618-622 (1992)). Activated PKC phosphorylates MARCKS,which is released from the membrane (Rosen et al, J. Exp. Med.,172:1211-1215 (1990); and Thelen et al, Nature, 351:320-322 (1991)). Theactin linked to MARCKS is likely to be spatially separated from themembrane and be more plastic. When MARCKS is dephosphorylated, itreturns to the membrane where it once again crosslinks actin (Hartwig etal, supra; and Thelen et al, supra). These data suggest that the F-actinnetwork may be rearranged by a PKC-dependent phosphorylation processthat involves actin-binding proteins (MARCKS being one of them).

A variety of intracellular mediators have been shown to alter tjfunction and/or structure. Tight junctions of amphibian gallbladder(Duffey et al, Nature, 204:451-452 (1981)), and both goldfish (Bakker etal, Am. J. Physiol., 246:G213-G217 (1984)) and flounder (Krasney at al,Fed. Proc., 42:1100 (1983)) intestine, display enhanced resistance topassive ion flow as intracellular cAMP is elevated. Also, exposure ofamphibian gallbladder to Ca²⁺ ionophore appears to enhance tjresistance, and induce alterations in tj structure (Palant et al, Am. J.Physiol., 245:C203-C212 (1983)). Further, activation of PKC by phorbolesters increases paracellular permeability both in kidney (Ellis at al,C. Am. J. Physiol., 263 (Renal Fluid Blectrolyte Physiol. 32):F293-F300(1992)), and intestinal (Stenson et al, C. Am. J. Physiol., 265(Gastrointet. Liver Physiol., 28):G955-G962 (1993)) epithelial celllines.

II. The Blood-Brain Barrier

The blood-brain barrier (BBB) is an extremely thin membranous barrierthat is highly resistant to solute free diffusion, and separates bloodand the brain. In molecular dimensions, the movement of drugs or solutethrough this membrane is essentially nil, unless the compound has accessto one of several specialized enzyme-like transport mechanisms that areembebbed within the BBB membrane. The BBB is composed of multiple cellsrather than a single layer of epithelial cells. Of the four differenttypes of cells that compose the BBB (endothelial cells, perycites,astrocytes, and neurons) the endothelial cell component of thecapillaries represents the limiting factor for the permeability of theBBB. The capillary endothelium in vertebrate brain and spinal cord isendowed with tj which closes the interendothelial pores that normallyexist in microvascular endothelial barriers in peripheral tissues.Ultimately, endotheial tj are responsible for the limited permeabilityof the BBB.

III. Zonula Occludens Toxin

Most Vibrio cholerae vaccine candidates constructed by deleting the ctxrgene encoding cholera toxin (CT) are able to elicit high antibodyresponses, but more than one-half of the vaccinees still develop milddiarrhea (Levine et al, Infect. Immun., 56(11:161-167 (1988)). Given themagnitude of the diarrhea induced in the absence of CT, it washypothesized that V. cholerae produce other enterotoxigenic factors,which are still present in strains deleted of the ctxA sequence (Levineet al, supra). As a result, a second toxin, zonula occludens toxin(hereinafter “ZOT”) elaborated by V. cholerae and which contribute tothe residual diarrhea, was discovered (Fasano et al, Proc. Natl. Acad.Sci., USA, 8:5242-5246 (1991)). The sot gene is located immediatelyadjacent to the ctx genes. The high percent concurrence of the sot genewith the ctx genes among V. cholerae strains (Johnson et al, J. Clin.Microb., 31/3:732-733 (1993); and Karasawa at al, FEB MicrobiologyLetters, 106:143-146 (1993)) suggests a possible synergistic role of ZOTin the causation of acute dehydrating diarrhea typical of cholera.Recently, the sot gene has also been identified in other entericpathogens (Tschape, 2nd Asian-Paciflc Symposium on Typhoid fever andother Salomellosis, 47(Abstr.) (1994)).

It has been previously found that, when tested on rabbit ileal rmucosa,ZOT increases the intestinal permeability by modulating the structure ofintercellular tj (Fasano et al, supra). It has been found that as aconsequence of modification of the paracellular pathway, the intestinalmucosa becomes more permeable. It also was found that ZOT does notaffect Na⁺-glucose coupled active transport, is not cytotoxic, and failsto completely abolish the transepithelial resistance (Fasano at al,supra).

More recently, it has been found that ZOT is capable of reversiblyopening tj in the intestinal mucosa, and thus ZOT, when co-administeredwith a therapeutic agent, is able to effect intestinal delivery of thetherapeutic agent, when employed in an oral dosage composition forintestinal drug delivery (WO 96/37196; U.S. patent application Ser. No.08/443,864, filed May 24, 1995; and U.S. Pat. No. 5,665,389; and Pasanoet al, J. Clin, Invest., 99:1158-1164 (1997); each of which isincorporated by reference herein in their entirety). It has also beenfound that ZOT is capable of reversibly opening tj in the nasal mucosa,and thus ZOT, when co-administered with a therapeutic agent, is able toenhance nasal absorption of a therapeutic agent (U.S. patent applicationSer. No. 08/781,057, filed Jan. 9, 1997; which is incorporated byreference herein in its entirety).

In U.S. patent application Ser. No. 08/803,364, filed Feb. 20, 1997;which is incorporated by reference herein in its entirety, a ZOTreceptor has been identified and purified from an intestinal cell line,i.e., CaCo2 cells. Further, in U.S. patent application Ser. No.09/024,198, filed Feb. 17, 1998; which is incorporated by referenceherein in its entirety, ZOT receptors from human intestinal, heart andbrain tissue have been identified and purified. The ZOT receptorsrepresent the first step of the paracellular pathway involved in theregulation of intestinal and nasal permeability.

IV. Zonulin

In pending U.S. patent application Ser. No. 098/859,931, filed May 21,1997, which is incorporated by reference herein in its entirety,mammalian proteins that are immunologically and functionally related toZOT, and that function as the physiological modulator of mammalian tightjunctions, have been identified and purified. These mammalian proteins,referred to as “zonulin”, are useful for enhancing absorption oftherapeutic agents across tj of intestinal and nasal mucosa, as well asacross tj of the blood brain barrier.

In the present invention, peptide antagonists of zonulin have beenidentified for the first time. Said peptide antagonists bind to ZOTreceptor, yet do not function to physiologically modulate the opening ofmammalian tight junctions. The peptide antagonists competitively inhibitthe binding of ZOT and zonulin to the ZOT receptor, thereby inhibitingthe ability of ZOT and zonulin to physiologically modulate the openingof mammalian tight junctions.

SUMMARY OF THE INVENTION

An object of the present invention is to identify peptide antagonists ofzonulin.

Another object of the present invention is to synthesize and purify saidpeptide antagonists.

Still another object of the present invention is to use said peptideantagonists as anti-inflammatory agents in the treatment ofgastrointestinal inflammation.

Yet another object of the present invention is to use said peptideantagonists to inhibit the breakdown of the blood brain barrier.

These and other objects of the present invention, which will be apparentfrom the detailed description of the invention provided hereinafter,have been met, in one embodiment, by a peptide antagonist of zonulincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID N10:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:35, wherein saidpeptide antagonist binds to a ZOT receptor, yet does not physiologicallymodulate the opening of mammalian tight junctions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of zonulin purified from rabbit intestine (▪),as compared to various negative controls (Fraction 2 (⋄); Fraction 3(); Fraction 4 (▴); and Fraction 5 (□) from a Q-Sepharose column), onthe tissue resistance (Rt) of CaCo2 cell monolayers.

FIG. 2 shows the effect of zonulin purified from rabbit intestine (▪),as compared to the negative control (□), on the tissue resistance (Rt)of rabbit ileum mounted in Ussing chambers.

FIG. 3 shows the effect of zonulin purified from rabbit intestine (▪),as compared to the negative controls (zonulin+anti-ZOT antibody (□);zonulin+anti-tau antibody (Δ); and tau (▴)), on the tissue resistance(Rt) of rabbit ileum mounted in Ussing chambers.

FIGS. 4A and 4B show the effect of zonulin purified from either humanbrain (▴), human intestine (), or human heart (∘), as compared to thenegative control (□), on the tissue resistance (Rt) of Rhesus monkeyjejunum (FIG. 4A) and Rhesus monkey ileum (FIG. 48) mounted in Ussingchambers.

FIGS. 5A and 5B show the effect of zonulin purified from either humanheart (▴) or human brain (□), as compared to the negative control (▪),on the tissue resistance (Rt) of rabbit jejunum (FIG. 5A) and rabbitileum (FIG. 5B) mounted in Ussing chambers.

FIG. 6 shows a comparison of the N-terminal sequence of zonulin purifiedfrom rabbit and various human tissues.

FIG. 7 shows a comparison of the N-terminal sequences of zonulinpurified from various human tissues and IgN heavy chain with theN-terminal sequence of the biologically active fragment (amino acids288-399) of ZOT.

FIG. 8 shows the effect of ZOT, zonulin, zonulin_(h), either alone(closed bars), or in combination with the peptide antagonist FZI/0 (openbars) or in combination with FZI/1 (shaded bars), as compared to thenegative control, on the tissue resistance (Rt) of rabbit ileum mountedin Ussing chambers. N equals 3-5; and * equals p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, in one embodiment, the above-described object of thepresent invention have been met by a peptide antagonist of zonulincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:35, wherein saidpeptide antagonist, binds to ZOT receptor, yet does not physiologicallymodulate the opening of mammalian tight junctions.

The size of the peptide antagonist is not critical to the presentinvention. Generally, the size of the peptide antagonist will range from8 to 110, amino acids, preferably from 8 to 40 amino acids, morepreferably will be 8 amino acids.

The peptide antagonists can be chemically synthesized and purified usingwell-known techniques, such as described in High Performance LiquidChromatography of Peptides and Proteins:Separation Analysis andConformation, Eds. Mant et al, C.R.C. Press (1991), and a peptidesynthesizer, such as Symphony (Protein Technologies, Inc); or by usingrecombinant DNA techniques, i.e., where the nucleotide sequence encodingthe peptide is inserted in an appropriate expression vector, e.g., an E.coli or yeast expression vector, expressed in the respective host cell,and purified therefrom using well-known techniques.

The peptide antagonists can be used as anti-inflammatory agents for thetreatment of gastrointestinal inflammation that gives rise to increasedintestinal permeability. Thus, the peptide antagonists of the presentinvention are useful, e.g., in the treatment of intestinal conditionsthat cause protein losing enteropathy. Protein losing enteropathy mayarise due to:

-   -   Infection, e.g., C. difficile infection, enterocolitis,        shigellosis, viral gastroenteritis, parasite infestation,        bacterial overgrowth, Whipple's disease;    -   Diseases with mucosal erosion or ulcerations, e.g., gastritis,        gastric cancer, collagenous colitis, inflammatory bowel disease;    -   Diseases marked by lymphatic obstruction, e.g., congenital        intestinal lymphangiectasia, sarcoidosis lymphoma, mesenteric        tuberculosis, and after surgical correction of congenital heart        disease with Fontan's operation;    -   Mucosal diseases without ulceration, e.g., Ménétrier's disease,        celiac disease, eosinophilic gastroenteritis; azid    -   Immune diseases, e.g., systemic lupus erythematosus or food        allergies, primarily to milk (see also Table 40-2 of Pediatric        Gastrointestinal Disease Pathophysiology Diagnosis Management,        Eds. Wyllie et al, Saunders Co. (1993), pages 536-543; which is        incorporated by reference herein in its entirety).

Hence, in another embodiment, the present invention relates to a methodfor treatment of gastrointestinal inflammation comprising administeringto a subject in need of such treatment, a pharmaceutically effectiveamount of a peptide antagonist of zonulin, wherein said peptideantagonist comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, wherein saidpeptide antagonist binds to the ZOT receptor in the intestine of saidsubject, yet does not physiologically modulate the opening of tightjunctions in said intestine.

To this end, the peptide antagonists can be administered as oral dosagecompositions for small intestinal delivery. Such oral dosagecompositions for small intestinal delivery are well-known in the art,and generally comprise gastroresistent tablets or capsules (Remington'sPharmaceutical Sciences, 16th Ed., Eds. Osol, Mack Publishing Co.,Chapter 89 (1980), Digenis et al, J. Pharm. Sci., 83:915-921 (1994);Vantini et al, Clinica Terapeutica, 145:445-451 (1993); Yoshitomi et al,Chem. Pharm. Bull., 40:1902-1905 (1992); Thoma et al, Pharmazie,46:331-336 (1991); Morishita et al, Drug Design and Delivery, 1:309-319(1991); and Lin et al, Pharmaceutical Res., 8:919-924 (1991)); each ofwhich is incorporated by reference herein in its entirety).

Tablets are made gastroresistent by the addition of, e.g., eithercellulose acetate phthalate or cellulose acetate terephthalate.

Capsules are solid dosage forms in which the peptide antagonist(s) isenclosed in either a hard or soft, soluble container or shell ofgelatin. The gelatin used in the manufacture of capsules is obtainedfrom collagenous material by hydrolysis. There are two types of gelatin.Type A, derived from pork skins by acid processing, and Type B, obtainedfrom bones and animal skins by alkaline processing. The use of hardgelatin capsules permit a choice in prescribing a single peptideantagonist or a combination thereof at the exact dosage level consideredbest for the individual subject. The hard gelatin capsule consists oftwo sections, one slipping over the other, thus completely surroundingthe peptide antagonist. These capsules are filled by introducing thepeptide antagonist, or gastroresistent beads containing the peptideantagonist, into the longer end of the capsule, and then slipping on thecap. Hard gelatin capsules are made largely from gelatin, FD&Ccolorants, and sometimes an opacifying agent, such as titanium dioxide.The USP permits the gelatin for this purpose to contain 0.15% (w/v)sulfur dioxide to prevent decomposition during manufacture.

In the context of the present invention, oral dosage compositions forsmall intestinal delivery also include liquid compositions which containaqueous buffering agents that prevent the peptide antagonist from beingsignificantly inactivated by gastric fluids in the stomach, therebyallowing the peptide antagonist to reach the small intestines in anactive form. Examples of such aqueous buffering agents which can beemployed in the present invention include bicarbonate buffer (pH 5.5 to8.7, preferably about pH 7.4).

When the oral dosage composition is a liquid composition, it ispreferable that the composition be prepared just prior to administrationso as to minimize stability problems. In this case, the liquidcomposition can be prepared by dissolving lyophilized peptide antagonistin the aqueous buffering agent.

The peptide antagonists can be used as to inhibit breakdown of the bloodbrain barrier. Thus, the peptide antagonists of the present inventionare useful, e.g., in the treatment of conditions associated withbreakdown of the blood brain barrier. Examples of such conditionsinclude osmotic injuries, e.g., cerebral ischemia, stroke or cerebraledema; hypertension; carbon dioxide; convulsive seizure; chemicaltoxins; uremia (renal insufficiency); meningitis, encephalitis,encephalomielitia, e.g., infective (viral (SRV, HIV, etc.), or bacterial(TB, H. influenzae, meningococcus, etc.) or allergic; tumors; traumaticbrain injuries; radiation brain injury; immaturity and kernicterus;demyelinating diseases, e.g., multiple sclerosis or Guillian-Barresyndrome.

Hence, in another embodiment, the present invention relates to a methodfor treatment of conditions associated with breakdown of the blood brainbarrier comprising administering to a subject in need of such treatment,a pharmaceutically effective amount of a peptide antagonist of zonulin,wherein said peptide antagonist comprises amino acid sequence SEQ IDNO:35, wherein said peptide antagonist binds to ZOT receptor in thebrain of said subject, yet does not physiologically modulate the openingof tight junctions in said brain.

To this end, the peptide antagonists can be administered as intravenousdosage compositions for delivery to the brain. Such compositions arewell-known in the art, and compositions generally comprise aphysiological diluent, e.g., distilled water, or 0.9% (w/v) NaCl.

The pharmaceutically effective amount of peptide antagonist employed isnot critical to the present invention and will vary depending upon thedisease or condition being treated, as well as the age, weight and sexof the subject being treated. Generally, the amount of peptideantagonist employed in the present invention to inhibit gastrointestinalinflammation or inhibit breakdown of the blood brain barrier, e.g., toinhibit zonulin biological activity, is in the range of about 7.5×10⁻⁶ Mto 7.5×10⁻³ M, preferably about 7.5×10⁻⁶ M to 7.5×10⁻⁴ M. To achievesuch a final concentration in, e.g., the intestines or blood, the amountof peptide antagonist in a single oral dosage composition of the presentinvention will generally be about 1.0 μg to 1000 μg, preferably about1.0 μg to 100 μg.

The peptide antagonists can be also be used as an immunogen to obtainantibodies, either polycolonal or monoclonal, having binding specificityfor zonulin, using techniques well-known in the art (Abrams, MethodsEnzymol., 21:107-119 (1986)). These antibodies can in turn can be usedto assay for zonulin in body tissue or fluids, or inaffinity-purification of zonulin, or alternatively, to bind to zonulin,and thereby inhibit zonulin activity, e.g., to inhibit gastrointestinalinflammation or to inhibit breakdown of the blood brain barrier.

The following examples are provided for illustrative purposes only, andare in no way intended to limit the scope of the present invention.

Example 1 Purification of ZOT

5000 μl of the supernatant fraction obtained after culturing V. choleraestrain CVD110 (Michalski et al, Infect. Imuan., G1:4462-4468 (1993),which had been transformed with plasmid pZ14, was concentrated 1000-foldusing a lamina flow filter with a NW cutoff of 10 kDa. The constructionof pZ14, which contains the vibrio cholera sot gene, is described indetail in, inter alia, MO 96/37196. The resulting supernatant was thensubjected to 8.0% (w/v) SDS-PAGE. Protein bands were detected byCoomassie blue staining of the SDS-PAGE gel. No protein bandcorresponding to ZOT was detectable when compared to control supernatantfrom strain CVD110 transformed with plasmid pTTQ181 (Amersham, ArlingtonHeights, Ill.), and treated in the same manner. Therefore, even thoughthe zot gene was placed behind the highly inducible and strong tacpromoter in pZ14, the level of the protein in 1000-fold concentratedpZ14 supernatant was still not detectable by the Coomassie stainedSDS-PAGE gel.

A. MBP-ZOT

To increase the amount of ZOT produced, the zot gene was fused in framewith the maltose binding protein (hereinafter “MBP”) gene to create aMBP-ZOT fusion protein.

The MBP vector pMAL-c2 (Biolab) was used to express and purify ZOT byfusing the sot gene to the male gene of E. coli. This construct uses thestrong, inducible tac promoter, and the malE translation initiationsignals to give high level expression of the cloned zot gene. The vectorpMAL-c2 has an exact deletion of the malE signal sequence, which leadsto cytoplasmic expression of the fusion protein. Affinity chromatographypurification for MBP was used to facilitate isolation of the fusionprotein (Biolab).

More specifically, vector pMAL-c2 was linearized with BcoRI (that cutsat the 3′ end of the malE gene), filled in with Klenow fragment, anddigested with XbaI (that has a single site in pAL-c2 polylinker). Theorf encoding ZOT was subcloned from plasmid pBB241 (Baudry et al,Infect. Immun., 60:428-434 (1992)). Plasamid pBB241 was digested withBsaHII, filled in with Klenow fragment, and digested with XhaI. Then,the blunt-XbaI fragment was subcloned into pMAL-c2 to give plasmidpLC10-c. Since both the insert, and the vector had blunt and stickyends, the correct orientation was obtained with the 3′ end of malE fusedwith the 5′ terminus of the insert. pLC10-c was then electroporated intoE. coli strain DH5α. In pBB241, the BamHII restriction site is withinthe sot orf. Thus, amino acids 1-8 of ZOT are missing in the MBP-ZOTfusion protein.

In order to purify the MBP-ZOT fusion protein, 10 ml of Luria Bertanibroth containing 0.2% (w/v) glucose and 100 μg/ml ampicillin wereinoculated with a single colony containing pLC10-c, and incubatedovernight at 37° C. with shaking. The culture was diluted 1:100 in 1.0ml of the same fresh medium, and grown at 37° C. while shaking, to about1.0×10⁸ cells/ml. 0.2 mM IPTG was then added to induce the MBP-ZOTexpression, and the culture was incubated at 37° C. for additional 3 hr.The bacteria were then pelleted and resuspended in 20 ml of ice cold“column buffer” comprising 20 mM Tris-HCl, 0.2 M NaCl, 1.0 mM EDTA, 10mM 2-ME, 1.0 mM NaN₃. The bacterial suspension was lysed by french presstreatment and spun for 30 min at 13,000×g at 4° C. The supernatant wascollected, diluted 1:5 with column buffer and loaded into a 1×10 columnof amylose resin (Biolabs, MBP-fusion purification system),pre-equilibrated with column buffer. After washing the column with 5volumes of column buffer, the MBP-ZOT fusion protein was eluted byloading 10 ml of 10 mM maltose in column buffer. The typical yield from1.0 ml of culture was 2-3 mg of protein.

The MBP fusion partner of the purified MBP-ZOT fusion protein was thencleaved off using 1.0 μg of Factor Xa protease (Biolabs) per 20 μg ofMBP-ZOT. Factor Xa protease cleaves just before the amino terminus ofZOT. The ZOT protein so obtained was run on a 8.0% (w/v) SDS-PAGE gel,and electroeluted from the gel using an electroseparation chamber(Schleicher & Schuell, Keene, N.H.).

When tested in Ussing chambers, the resulting purified ZOT induced adose-dependent decrease of Rt, with an ED₅₀ of 7.5×10⁻⁸ M.

B. 6×His-ZOT

The zot gene was amplified by PCR with Deep Vent polymerase (New EnglandBiolabs), using pBB241 plasmid (Baudry et al, supra) DNA as a template.The forward and reverse primers used were: 5′-CGGATCCCGTATGAGTATCTTT-3′(SEQ ID NO:39); and 5′-CCCAAGCTTGGGTCAAAATATACT-3′ (SEQ ID NO:40),respectively. The 5′ tails of these oligonucleotides contain a BamHI anda HindIII restriction site, respectively. The resulting amplicon (1.2kb) was analyzed by 8.0% (w/v) agarose gel electrophoresis, and purifiedfrom salts and free nucleotides using an Xtreme spin column (Pierce).The above-noted two restriction enzymes were then used to digest thepurified amplicon, and the resulting digested-amplicon was then insertedin the vector pQE30 (Quiagen), which had been previously digested withBamHI and HindIII, so as to obtain plasmid pSU113. pQE30 is anexpression vector that provides high level expression of a recombinantprotein with a 6 poly-histidine tag (6×His). The expression product ofplasmid pSU113 is therefore a 6×His-ZOT fusion protein. pSU113 was thentransformed into E. coli DH5α.

In order to purify the 6×His-ZOT fusion protein, the resultingtransformed E. coli were grown overnight at 37° C. in 150 ml of LuriaBertani broth containing 2.0% (w/v) glucose, 25 μg/ml of kanamycin and200 μg/ml of ampicillin until the A₆₀₀ was about 1.10. Next, 75 ml ofthe overnight cultures were added to 1000 ml of Luria Bertani brothcontaining 2.0% (w/v) glucose, 25 μg/ml of kanamycin and 200 μg/ml ofampicillin, incubated for about 3 hrs at 37° C., with vigorous shaking,until the A₆₀₀ was about 0.7-0.9. Then, IPTG was added to a finalconcentration of 2.0 mM, and growth was allowed to continue for 5 hrs at37° C. Next, the cells were harvested by centrifugation at 4000×g for 20min, the cells resuspend in 5.0 ml/g wet weight of buffer A comprising6.0 M GuHCl, 0.1 M sodium phosphate, and 0.01 M Tris-HCl (pH 8.0), andstirred for 1 hr at room temperature. Then, the mixture was centrifugedat 10,000×g for 30 min at 4° C., and to the resulting supernatant wasadded 4.0-5.0 ml/g wet weight of a 50% slurry of SUPERFLOW resin(QIAGEN), and stirring was carried out for 1 hr at room temperature. Theresulting resin was loaded into a 1.6×8.0 column, which was then washedsequentially with buffer A, buffer B comprising 8.0 M urea, 0.1 M sodiumphosphate, and 0.01 M Tris-HCl (pH 8.0) and buffer C comprising 8.0 Murea, 0.1 M sodium phosphate, and 0.01 M Tris-HCl (pH 6.3). Each washwas carried out until the A₆₀₀ of the flow-through was less than 0.01.The 6×His-ZOT fusion protein was eluted from the column using 20 ml ofbuffer C containing 250 mM imidazole. Then, the fractions containingwith the 6×His-ZOT fusion protein were checked by SDS-PAGH using theprocedure described by Davis, Ann. N.Y. Acad. Sci., 121: 404 (1964), andthe gel stained with Comassie blue. The fractions containing 6×His-ZOTfusion protein were dialyzed against 8.0 n urea, combined, and thendiluted 100 times in PBS. Next, 4.0 ml of a 50% slurry of SUPERFLOWresin was added, stirring was carried out for 2 hrs at room temperature,and the resulting resin loaded into a 1.6×8.0 column, which was thenwashed with 50 ml of PBS. The 6×His-ZOT fusion protein was eluted fromthe column with 10 ml of PBS containing 250 mM imidazole. The resultingeluant was dialyzed against PBS, and the 6×His-ZOT fusion protein waschecked by SDS-PAGE, as described above.

Example 2 Production of Affinity-Purified Anti-ZOT Antibodies

To obtain specific antiserum, a chimeric glutathione S-transferase(GST)-ZOT protein was expressed and purified.

More specifically, oligonucleotide primers were used to amplify the zotorf by polymerase chain reaction (PCR) using plasmid pBB241 (Baudry etal, supra) as template DNA. The forward primer (TCATCACGGC GCGCCAGG, SEQID NO:25) corresponded to nucleotides 15-32 of zot orf, and the reverseprimer (GGAGGTCTAG AATCTGCCCG AT, SEQ ID NO:26) corresponded to the 5′end of ctxA orf. Therefore, amino acids 1-5 of ZOT were missing in theresulting fusion protein. The amplification product was inserted intothe polylinker (SmaI site) located at the end of the GST gene in pGEX-2T(Pharmacia, Milwaukee, Wis.), pGEX-2T is a fusion-protein expressionvector that expresses a cloned gene as a fusion protein with GST ofSchistosoma japonicum. The fusion gene is under the control of the tacpromoter. Upon induction with IPTG, derepression occurs and GST fusionprotein is expressed.

The resulting recombinant plasmid, named pLC11, was electroporated in E.coli DH5α. In order to purify GST-ZOT fusion protein, 10 ml of LuriaBertani broth containing 100 μg/ml ampicillin were inoculated with asingle colony containing pLC11, and incubated overnight at 37° C. withshaking. The culture was diluted 1:100 in 1.0 ml of the same freshmedium and grown at 37° C. while shaking, to about 1.0×10⁸ cells/ml. 0.2mM IPTG was then added to induce the GST-ZOT expression, and the culturewas incubated at 37° C. for additional 3 hr. The bacteria were thenpelleted, resuspended in 20 ml of ice cold PBS (pH 7.4), and lysed bythe french press method. The GST-ZOT fusion protein was not solubleunder these conditions as it sedimented with the bacterial pelletfraction. Therefore, the pellet was resuspended in Laemli lysis buffercomprising 0.00625 M Tris-HCl (pH 6.8), 0.2 M 2-ME, 2.0% (w/v) SDS,0.025% (w/v) bromophenol blue and 10% (v/v) glycerol, and subjected toelectrophoresis on a 8.0% (w/v) PAGE-SDS gel, and stained with Coomassiebrilliant blue. A band of about 70 kDa (26 kDa of GST+44 kDA of ZOT),corresponding to the fusion protein, was electroeluted from the gelusing an electroseparation chamber (Schleicher & Schuell, Keene, N.H.).

10 μg of the resulting eluted protein (10-20 μg) was injected into arabbit mixed with an equal volume of Freund's complete adjuvant. Twobooster doses were administered with Freund's incomplete adjuvant fourand eight weeks later. One month later the rabbit was bled.

To determine the production of specific antibodies, 10⁻¹⁰ M of ZOT,along with the two fusion proteins MBP-ZOT and GST-ZOT, was transferredonto a nylon membrane and incubated with a 1:5000 dilution of the rabbitantiserum overnight at 4° C. with moderate shaking. The filter was thenwashed 15 min 4 times with PBS containing 0.05% (v/v) Tween 20(hereinafter “PBS-T”), and incubated with a 1:30,000 dilution of goatanti-rabbit IgG conjugated to horseradish peroxidase for 2 hr at roomtemperature. The filter was washed again for 15 min 4 times with PBScontaining 0.1% (v/v) Tween, and immunoreactive bands were detectedusing enhanced chemiluminescence (Amersham).

On immunoblot, the rabbit antiserum was found to recognize ZOT, as wellas MBP-ZOT and GST-ZOT fusion proteins, but not the MBP negativecontrol.

Moreover, to confirm the production of appropriate anti-ZOT antibodies,neutralization experiments were conducted in Ussing chambers. Whenpre-incubated with pZ14 supernatant at 37° C. for 60 min, theZOT-specific antiserum (1:100 dilution), was able to completelyneutralize the decrease in Rt induced by ZOT on rabbit ileum mounted inUssing chambers.

Next, the anti-ZOT antibodies were affinity-purified using an MBP-ZOTaffinity column. More specifically, a MBP-ZOT affinity column wasprepared by immobilizing, overnight at room temperature, 1.0 mg ofpurified MBP-ZOT, obtained as described in Example 1 above, to apre-activated gel (Aminolink, Pierce). The column was washed with PBS,and then loaded with 2.0 ml of anti-ZOT rabbit antiserum. After a 90 minincubation at room temperature, the column was washed with 14 ml of PBS,and the specific anti-ZOT antibodies were eluted from the column with4.0 ml of a solution comprising 50 mM glycine (pH 2.5), 150 mM NaCl, and0.1% (v/v) Triton X-100. The pH of the 1.0 ml eluted fractions wasimmediately neutralized with 1.0 N NaOH.

Example 3 Purification of Zonulin

Based upon the observation in U.S. patent application Ser. No.08/803,364, filed Feb. 20, 1997, that ZOT interacts with a specificepithelial surface receptor, with subsequent activation of a complexintracellular cascade of events that regulate tj permeability, it waspostulated in the present invention that ZOT may mimic the effect of aphysiological modulator of mammalian tj. It was postulated in U.S.patent application Ser. No. 08/859,931, filed May 21, 1997, that ZOT,and its physiological analog (zonulin), would be functionally andimmunologically related. Therefore, as described therein,affinity-purified anti-ZOT antibodies and the Ussing chamber assay wereused in combination to search for zonulin in various rabbit and humantissues.

A. Rabbit Tissues

Initially, zonulin was purified from rabbit intestine. The tissue wasdisrupted by homogenization in PBS. The resulting cell preparations werethan centrifuged at 40,000 rpm for 30 min, the supernatant collected andlyophilized. The resulting lyophilized product was subsequentlyreconstituted in PBS (10:1 (v/v)), filtered through a 0.45 mm membranefilter, loaded onto a Sephadex 0-50 chromatographic column, and elutedwith PBS. Then, 2.0 ml fractions obtained from the column were subjectedto standard Western immunoblotting using the affinity-purified anti-ZOTantibodies obtained as described in Example 2 above.

Positive fractions, i.e., those to which the anti-ZOT antibodies bound,were combined, lyophilized, reconstituted in PBS (1:1 (v/v)), andsubjected to salt gradient chromatography through a Q-Sepharose column.The salt gradient was 0-100% (w/v) NaCl in 50 mM Tris buffer (pH 8.0).Five 20 ml fractions were collected, and subjected to standard Westernimmunoblotting using the affinity-purified anti-ZOT antibodies obtainedas described in Example 2 above. Fraction 1 (20% (w/v) NaCl) was theonly fraction that was found to be positive in the Western immunoblotassay.

The fractions obtained from the Q-Sepharose column were then tested fortheir tissue resistance effects on both CaCo2 monolayers, and rabbitsmall intestine in Ussing chambers.

More specifically, CaCo2 cells were grown in cell-culture flasks(Falcon) under humidified atmosphere of 95% O₂/5% CO₂ at 37° C. inDulbecco's modified Eagle's medium containing 10% (v/v) fetal-calfserum, 40 μg/l penicillin and 90 μg/l streptomycin. The cells weresubcultured at a surface ratio of 1:5 after trypsin treatment every 5days, when they had reached 70-80% confluence. The passage number of thecells used in the this study varied between 15 and 30.

The CaCo2 monolayers were grown to confluence (12-14 days after platingat a 1:2.5 surface ratio) on tissue-culture-treated polycarbonatefilters firmly attached to a polystyrene ring (6.4 mm diameter,Transwell Costar). The filters were placed in a tightly fitting insertseparating the serosal and mucosal compartment of a modified Ussingchamber, and the experiments were carried out as described by Fasano etal, Proc. Natl. Acad. Sci., USA, 8:5242-5246 (1991), for rabbitintestines in Ussing chambers. The results are shown in FIG. 1.

As shown in FIG. 1, the zonulin-containing fraction induced asignificant reduction of CaCo2 monolayers' resistance, as compared tozonulin-negative fractions.

Next, Ussing chamber assays were carried out using ileum from 2-3 kgadult male New Zealand white rabbits, which were sacrificed by cervicaldislocation. A 20 cm segment of ileum was removed, rinsed free of theintestinal content, opened along the mesenteric border, and stripped ofmuscular and serosal layers. Eight sheets of mucosa so prepared werethen mounted in lucite Ussing chambers (1.12 cm² opening), connected toa voltage clamp apparatus (EVC 4000 WPI, Saratosa, Fla.), and bathedwith freshly prepared Ringer's solution comprising 53 mM NaCl, 5.0 mMKCl, 30.5 sa mannitol, 1.69 mM Na₂HPO₄, 0.3 mM NaH₂PO₄, 1.25 mM CaCl₂,1.1 mM MgCl₂, and 25 mM NaHCO₃. The bathing solution was maintained at37° C. with water-jacketed reservoirs connected to aconstant-temperature circulating pump and gassed with 95% O₂/5% CO₂.

100 μl of zonulin purified from rabbit intestine was added to themucosal side. The potential difference (PD) was measured every 10 min,and the short-circuit current (Isc) and tissue resistance (Rt) werecalculated as described by Fasano et al, supra. Because of tissuevariability, data were calculated as ΔRt (Rt at time x)−(Rt at time 0).The results are shown in FIG. 2.

As shown in FIG. 2, the zonulin-containing traction induced asignificant reduction in rabbit small intestinal resistance, as comparedto a zonulin-negative fraction. This effect was completely reversibleonce zonulin was withdrawn from the reservoir.

The zonulin-positive fraction was also subjected to 8.0% (w/v) SDS-PAGE,followed by Western immunoblotting using the anti-ZOT antibodies. Theprotein bands separated by SDS-PAGE were then transferred onto PVDFfilter (Millipore) using CAPS buffer comprising 100 ml of(3-[cyclohexylamino]-1 propanesulfonic acid) 10×, 100 ml of methanol,800 ml of distilled water. The protein that aligned to a single bandthat was detected by Western immunoblotting had an apparent molecularweight of about 47 kDa. This band was cut out from the PVDF filter, andsubjected to N-terminal sequencing as described by Hunkapiller, In:Methods of Protein Microcharacterization, Ed. Shibley, Chapters 11-12,Humana Press, pages 315-334 (1985), using a Perkin-Elmer AppliedBiosystems Apparatus Model 494. The N-terminal sequence of zonulinpurified from rabbit intestine is shown in SEQ ID NO:27.

The rabbit zonulin N-terminal sequence was compared to other proteinsequences by BLAST search analysis. The result of this analysis revealedthat the N-terminal sequence of rabbit zonulin is 85% identical, and100% similar, to the N-terminal sequence of tau protein from Homosapiens.

As a result, to determine whether rabbit zonulin and tau are the samemoiety, cross-neutralization experiments were conducted in Ussingchambers. More specifically, 10 μl/ml of rabbit zonulin was added to themucosal aide of rabbit ileum either untreated or pre-incubated for 60min at 37° C. with anti-tau antibodies (dilution 1:10) (Sigma). Both 10μl/ml of rabbit zonulin pre-incubated with anti-ZOT antibodies (dilution1:10) (Example 2); and 0.4 μg/ml of purified tau (Sigma), were used ascontrols. The results are shown in FIG. 3.

As shown in FIG. 3, rabbit zonulin induced the typical decrease oftissue resistance that was readily reversible once the protein waswithdrawn from the Ussing chambers. This activity was completelyneutralized by pre-treatment with anti-ZOT antibodies, but not bypre-treatment with anti-tau antibodies. On the other hand, there was nosignificant effect on tissue resistance in tissues exposed to tauprotein.

Rabbit zonulin was also detected in various other rabbit tissues, i.e.,rabbit heart, brain, muscle, stomach, spleen, lung, kidney, as well asvarious portions of rabbits intestines, i.e., distal jejunum, proximaljejunum, ileum, caecum and colon. That is, when these rabbit tissueswere processed in the same manner as the rabbit intestine, discussedabove, and subjected to 8.0% (w/v) SDS-PAGE, followed by Westernimmunoblotting using affinity-purified anti-ZOT antibodies obtained asdescribed in Example 2 above, a single band of approximately 47 kDa insize was detected in all of the tissues tested.

B. Human Tissues

Zonulin was also purified from several human tissues, includingintestine, heart, and brain. Both fetal and adult tissues were used. Thetissues were disrupted by homogenization in PBS. The resulting cellpreparations were than centrifuged at 40,000 rpm for 30 min, thesupernatant collected and lyophilized. The resulting lyophilized productwas subsequently reconstituted in PBS (10:1 (v/v)), filtered through a0.45 mm membrane filter, loaded onto a Sephadex G-50 chromatographiccolumn, and eluted with PBS. Then, 2.0 ml fractions obtained from thecolumn were subjected to standard Western immunoblotting using theaffinity-purified anti-ZOT antibodies obtained as described in Example 2above.

Positive fractions, i.e., those to which the anti-ZOT antibodies bound,were combined, lyophilized, reconstituted in PBS (1:1 (v/v)), andsubjected to salt gradient chromatography through a Q-Sepharose column.The salt gradient was 0-100% (w/v) NaCl in 50 mM Tris buffer (pH 7.4).Five 20 ml fractions were collected, and subjected to standard Westernimmunoblotting using the affinity-purified anti-ZOT antibodies obtainedas described in Example 2 above. Fraction 1 (20% (w/v) NaCl) showed asingle band of 47 kDa in size in the Western immunoblot assay. Fraction2 (40% (w/v) NaCl) showed two additional bands of 35 kDa and 15 kDa insize in the Western immunoblot assay. Fraction 3 (60% (w/v) NaCl) andFraction 4 (80% (w/v) showed only the 35 kDa and 15 kDa bands. Theseresults suggest that zonulin may be subjected to degradation byproteases, probably present in the human tissues used, and that thebreakdown products elute from the column at higher salt concentrationsas compared to the holoprotein.

Fraction 1 (from human heart, intestine and brain tissues) and Fraction4 (from heart tissue) obtained from the Q-Sepharose column were thentested for their tissue resistance effects on both rabbit intestine andRhesus monkey intestine in Ussing chambers.

Ussing chamber assays were carried out using different tracts ofintestine, including jejunum, ileum, or colon from either 2-3 kg adultmale New Zealand white rabbits, or 5-6 kg adult male Rhesus monkeys.After the animals were sacrificed, different segments of intestine,including jejunum, ileum, and colon, were removed, rinsed free of theintestinal content, opened along the mesenteric border, and stripped ofmuscular and serosal layers. Bight sheets of mucosa so prepared (threejejunum, three ileum, and two colon) were then mounted in lucite Ussingchambers (1.12 cm² opening), connected to a voltage clamp apparatus (EVC4000 WPI, Saratosa, Fla.), and bathed with freshly prepared Ringer'ssolution comprising 53 mM NaCl, 5.0 mM KCl, 30.5 mM mannitol, 1.69 mMNa₂HPO₄, 0.3 mM NaH₂PO₄, 1.25 ma CaCl₂, 1.1 mM MgCl₂, and 25 mM NaHCO₃.The bathing solution was maintained at 37° C. with water-jacketedreservoirs connected to a constant-temperature circulating pump andgassed with 95% O₂/5% CO₂.

100 μl of Fraction 1 of zonulin purified from human heart or Fraction 1of zonulin purified from human brain, or Fraction 1 of zonulin purifiedfrom human intestine, or Fraction 4 purified from human heart, was addedto the mucosal side. The potential difference (PD) was measured every 10min, and the short-circuit current (Isc) and tissue resistance (Rt) werecalculated as described by Fasano et al, supra. Data were calculated asRt for FIGS. 4A and 4B; but because of tissue variability, data werecalculated as ΔRt (Rt at time x)−(Rt at time 0) for FIGS. 5A and 5B. Theresults are shown in FIGS. 4A and 4B (monkey intestine) and FIGS. 5A and5B (rabbit intestine).

As shown in FIGS. 4A and 4B, zonulin purified from human heart andintestine (Fraction 1) induced a significant reduction in monkeyintestinal resistance (both jejunum (FIG. 4A) and ileum (FIG. 48), ascompared to the PBS negative control. No significant changes wereobserved when zonulin purified from either human heart or humanintestine were tested in the colon. FIGS. 4A and 4B also show that nosignificant effect on both monkey jejunum (FIG. 4A) and monkey ileum(FIG. 4B) was observed when zonulin purified from human brain(Fraction 1) was tested. Fraction 4 of zonulin purified from human heartalso induced a significant decrease in monkey small intestinal tissueresistance.

As shown in FIGS. 5A and 5B, similar results were obtained when rabbitintestine was used. That is, zonulin purified from human heart(Fraction 1) showed a significant effect on tissue resistance both inthe rabbit jejunum (FIG. 5A) and rabbit ileum (FIG. 5B), but not in thecolon. FIGS. 5A and 5B also show that no significant effect on bothrabbit jejunum (FIG. 5A) and rabbit ileum (FIG. 5B) was observed whenzonulin purified from human brain (Fraction 1) was tested.

To establish whether zonulin increases the oral delivery of insulin, invitro model experiments using rabbit intestine were performed. Briefly,adult male New Zealand white rabbits (2-3 kg) were sacrificed bycervical dislocation. Segments of rabbit small intestine (either jejunumor ileum) were removed, rinsed free of the intestinal content, openedalong the mesenteric border, and stripped of muscular and serosallayers. Bight sheets of mucosa so prepared were then mounted in LuciteUssing chambers (1.12 cm² opening), connected to a voltage clampapparatus (EVC 4000 WPI, Sarasota, Fla.), and bathed with freshlyprepared buffer containing 53 N NaCl, 5.0 mM KCl, 30.5 mM mannitol, 1.69mM Na₂PO₄, 0.3 mM NaH₂PO₄, 1.25 mM CaCl₂, 1.1 mM MgCl₂, and 25 mMNaHCO₃. The bathing solution was maintained at 37° C. withwater-jacketed reservoirs connected to a constant-temperaturecirculating pump and gassed with 95% O₂/5% CO₂. Potential difference(PD) was measured, and short-circuit current (Isc) and tissue resistance(Rt) were calculated. Once the tissues reached a steady state condition,paired tissues, matched on the basis of their resistance, were exposedluminally to 10⁻¹¹ M ¹²⁵I-insulin (Amersham, Arlington Heights, Ill.;2.0 μCi=10⁻¹² M), alone or in the presence of 100 μl of heart zonulinfrom Fraction 1. A 1.0 ml aliquot from the serosal side and a 50 μlaliquot from the mucosal side were immediately obtained to establishbaseline values. Samples from the serosal side were then collected at 20min intervals for the following 100 min.

It was found that heart zonulin increased the intestinal absorption ofinsulin both in the jejunum (0.058±0.003 fmol/cm⁻²·min vs. 0.12±0.005fmol/cm⁻²·min, untreated vs. zonulin-treated tissues, respectively,p=0.001), and in the ileum (0.006±0.0002 fmol/cm²·min vs. 0.018±0.005fmol/cm²·min, untreated vs. zonulin-treated tissues, respectively,p=0.05) in a time-dependent manner.

Fraction 1 of zonulin purified from human heart, Fraction 1 of zonulinpurified from human intestine, and Fraction 1 of zonulin purified fromhuman brain were also subjected to 8.0% (w/v) SDS-PAGE, followed byWestern immunoblotting using the anti-ZOT antibodies obtained asdescribed in Example 2 above.

The protein bands separated by SDS-PAGE were then transferred onto PVDFfilter using CAPS buffer comprising 100 ml of (3-[cyclohexylamino]-1propanesulfonic acid) 10×, 100 ml of methanol, 800 ml of distilledwater. The protein that aligned to a single band that was detected byWestern immunoblotting had an apparent molecular weight of about 47 kDa.This band was cut out from the PDVF filter, and subjected to N-terminalsequencing as described by unkapiller, In: Methods of ProteinMicrocharacterization, Ed. Shibley, Chapters 11-12, Humana Press, pages315-334 (1985), using a Perkin-Elmer Applied Biosystems Apparatus Model494. The N-terminal sequence of zonulin purified from adult human heartis shown in SEQ ID NO:28, the N-terminal sequence of zonulin purifiedfrom adult human brain is shown in SEQ ID NO:29, and the N-terminalsequence of zonulin purified from adult fetal brain is shown in SEQ IDNO:36.

The first nine amino acids from the N-terminal sequence of zonulinpurified from adult human intestine (SEQ ID NO:31) were also sequenced,and found to be identical to the first nine amino acids of zonulinpurified from human heart shown in SEQ ID NO:28 (see FIG. 6). The firsttwenty amino acids from the N-terminal sequence of zonulin purified fromhuman fetal intestine were also sequenced: Met Leu Gln Lys Ala Glu SerGly Gly Val Leu Val Gln Pro Gly Xaa Ser Asn Arg Leu (SEQ ID NO:30), andfound to be almost identical to the amino acid sequence of zonulinpurified from human heart shown in SEQ ID NO:28 (see FIG. 6).

The N-terminal sequence of zonulin purified from adult human brain (SEQID NO:29) and fetal human brain (SEQ ID NO:36) was completely differentthan the N-terminal of zonulin purified from each of heart (SEQ IDNO:28), fetal intestine (SEQ ID NO:30) and adult intestine (SEQ IDNO:31) (see FIGS. 6-7). This difference is believed to explain thetissue-specificity of zonulin in determining the permeability oftissues, such as the intestine, demonstrated above.

The N-terminal sequences of human zonulin purified from heart,intestine, and brain, all differ from the N-terminal sequence of zonulinpurified from rabbit intestine (FIG. 6). To establish whether theseproteins represent different isoforms of a tau-related family ofproteins, tissues from both rabbit and human were subjected to 8.0%(w/v) SDS-PAGE, followed by Western immunoblotting using either anti-ZOTor anti-tau antibodies. The 47 kDa zonulin bands purified from bothrabbit and human tissues (including brain, intestine, and heart) whichwere found to be recognized by the anti-ZOT antibodies, were also foundto cross-react with anti-tau antibodies. The different fractions ofzonulin purified from human brain obtained by salt chromatography werealso subjected to Western immunoblotting using either anti-ZOTantibodies or anti-tau antibodies. Whiles anti-ZOT antibodies recognizedthe intact 47 kDa protein and both of the 35 kDa and 15 kDa breakdownfragments, the anti-tau antibodies only recognized the intact 47 kDaprotein and the 35 kDa fragment, while the anti-tau antibodies did notrecognize the 15 kDa fragment. To establish whether the 35 kDa fragmentincludes the N-terminus or the C-terminus of zonulin, the N-terminalsequence of the 35 kDa band was obtained and found to be: Xaa Xaa AspGly Thr Gly Lye Val Gly Asp Leu (SEQ ID NO:32). This sequence isdifferent from the N-terminal sequence of the intact human brain zonulin(830 ID NO:29). These results suggest that the 15 kDa fragmentrepresents the N-terminal portion of zonulin, while the 35 kDa fragmentrepresents the C-terminal portion of zonulin.

Combined together, these results suggest that the zonulin domainrecognized by the anti-tau antibodies is toward the C-terminus of theprotein, is common to the different isoforms of zonulin from eitherhuman or rabbit tissues (while the N-terminal portion may vary), and isprobably involved in the permeabilizing effect of the protein (based onthe observation that tau binds to β-tubulin with subsequentrearrangement of the cell cytoskeleton, and the effect of Fraction 4 onmonkey small intestinal tissue resistance).

The N-terminal sequence of human zonulin purified from both the heartand intestine was compared to other protein sequences by BLAST searchanalysis. The result of this analysis revealed that the N-terminalsequence of human zonulin is 95% identical, to the N-terminal sequenceof the heavy variable chain of IgM from Homo sapiens (SEQ ID NO:37).

As a result, to determine whether human zonulin purified from heart andhuman IgM are the same moiety, a partial digestion of the human zonulinwas performed to obtain an internal fragment, which was then sequenced.

More specifically, 1.0 mm of the PVDF filter containing zonulin purifiedfrom human heart was placed in a plastic tube previously washed with0.1% (w/v) trifluoracetic acid (TFA), and rinsed with methanol. 75 μl ofa buffer solution comprising 100 mM Tris (pH 8.2), 10% (v/v) CH₂CN, and1.0% (v/v) dehydrogenated Triton X-100 was added, and incubated with themembrane at 37° C. for 60 min. 150 ng of trypsin was then added, and anadditional 24 hr incubation period at 37° C. was carried out. Theresulting solution was sonicated for 10 min, and the supernatantdecanted. 75 μl of 0.1% (w/v) TFA was then added, the solution wassonicated for additional 10 min, and the supernatant decanted. Bothaliquots were loaded on a 0.5 mm×250 mm C₁₈ column, 5.0 μm particlesize, 300 Å pore size. A gradient from 0.1% (w/v) TFA to 45% CH₃CNwater+0.1% (w/v) TFA was developed for 2 hr and 15 min. The peaks werefinally collected and sequenced.

The internal sequence of human zonulin purified from adult human heartwas found to be: Leu Ser Glu Val Thr Ala Val Pro Ser Leu Asn Gly Gly(SEQ ID NO:33).

The human zonulin internal sequence was compared to other proteinsequences by BLAST search analysis. The result of this analysis revealedthat the internal sequence of human zonulin has 0% identity to anyinternal sequence of the heavy variable chain of IgM from Homo sapiens.

The results in Example 3 above demonstrate that (1) zonulin representsthe physiological modulator of the paracellular pathway; (2) theN-terminal sequence of rabbit zonulin is highly homologous to theN-terminal sequence of the tau protein; (3) zonulin and tau are twodistinct moieties that are immunologically related, yet functionallydifferent; (4) the N-terminal sequence of human zonulin obtained fromheart and intestine is highly homologous to the N-terminal sequence ofthe heavy chain of the variable region of IgM; (5) human zonulin and IgMare two distinct moieties that are structurally related, yetfunctionally different; and (6) zonulin represents a family oftau-related proteins with common, active C-terminal sequences, andvariable N-terminal sequences.

Example 4 Peptide Antagonists of Zonulin

Given that ZOT, human intestinal zonulin (zonulin_(i)) and human heartzonulin (zonulin) all act on intestinal (Fasano et al, Gastroenterology,1:839 (1997); Fasano et al, J. Clin. Invest., 296710 (1995); and FIGS.1-5) and endothelial tj and that all three have a similar regionaleffect (Fasano et al (1997), supra; and FIGS. 1-5) that coincides withthe ZOT receptor distribution within the intestine (Fasano et al (1997),supra; and Fasano et al (1995), supra), it was postulated in the presentinvention that these three molecules interact with the same receptorbinding site. A comparison of the primary amino acid structure of ZOTand the human zonulins was thus carried out to provide insights as tothe absolute structural requirements of the receptor-ligand interactioninvolved in the regulation of intestinal tj. The analysis of theN-termini of these molecules revealed the following common motif (aminoacid residues 8-15 boxed in FIG. 7): non-polar (Gly for intestine, Valfor brain), variable, non-polar, variable, non-polar, polar, variable,polar (Gly). Gly in position 8, Val in position 12 and Gln in position13, all are highly conserved in ZOT, zonulin_(i) and zonulin, (see FIG.7), which is believed to be critical for receptor binding functionwithin the intestine. To verify the same, the synthetic octapeptide GlyGly Val Leu Val Gln Pro Gly (SEQ ID NO:15) (named FZI/0, andcorresponding to amino acid residues 8-15 of human fetal zonulins) waschemically synthesized.

Next, rabbit ileum mounted in Ussing chambers as described above, wereexposed to 100 μg of FZI/0 (SEQ ID NO:15), 100 μg of FZI/1 (SEQ IDNO:34), 1.0 μg of 6×His-ZOT (obtained as described in Example 1), 1.0 μgof zonulin_(i) (obtained as described in Example 3), or 1.0 μg ofzonulin_(h) (obtained as described in Example 3) alone; or pre-exposedfor 20 min to 100 μg of FZI/0 or FZI/1, at which time 1.0 μg of6×His-ZOT, 1.0 μg of zonulin, or 1.0 μg of zonulin_(h), was added. ΔRtwas then calculated as described above. The results are shown in FIG. 8.

As shown in FIG. 8, FZI/0 did not induce any significant change in Rt(0.5% as compared to the negative control) (see closed bar). On thecontrary, pre-treatment for 20 min with FZI/0 decreased the effect ofZOT, zonulin, and zonulin_(h) on Rt by 75%, 97%, and 100%, respectively(see open bar). Also as shown in FIG. 8, this inhibitory effect wascompletely ablated when a second synthetic peptide (FZI/1) waschemically synthesized by changing the Gly in position 8, the Val inposition 12, and the Gln in position 13 (as referred to zonulin_(i))with the correspondent amino acid residues of zonulin, (Val, Gly, andArg, respectively) was used (see shaded bar).

The above results demonstrate that there is a region spanning betweenresidue 8 and 15 of the N-terminal end of ZOT and the zonulin familythat is crucial for the binding to the target receptor, and that theamino acid residues in position 8, 12, and 13 determine the tissuespecificity of this binding.

While the invention has been described in detail, and with reference tospecific embodiments thereof, it will be apparent to one of ordinaryskill in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

What is claimed is:
 1. A method for treatment of a condition associatedwith breakdown of the blood brain barrier comprising administering to asubject in need of such treatment, a pharmaceutically effective amountof a peptide antagonist of zonulin, wherein the peptide antagonistcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:35; and whereinthe peptide antagonist binds to a zonula occludens toxin receptor, yetdoes not physiologically modulate the opening of mammalian tightjunctions.
 2. The method of claim 1, wherein the zonula occludens toxinreceptor is in brain tissue.
 3. The method of claim 1, wherein thepeptide competitively inhibits zonula occludens toxin (ZOT) and zonulinfrom binding to the zonula occludens toxin receptor and from modulatingthe opening of the mammalian tight junction.
 4. The method of claim 1,wherein the condition is cerebral ischemia.
 5. The method of claim 1,wherein the condition is stroke or cerebral edema.
 6. The method ofclaim 1, wherein the condition is hypertension.
 7. The method of claim1, wherein the condition is convulsive seizure.
 8. The method of claim1, wherein the condition is uremia.
 9. The method of claim 1, whereinthe condition is meningitis.
 10. The method of claim 1, wherein thecondition is encephalitis.
 11. The method of claim 1, wherein thecondition is encephalomyelitis.
 12. The method of claim 1, wherein thecondition is traumatic brain injury.
 13. The method of claim 1, whereinthe condition is radiation brain injury.
 14. The method of claim 1,wherein the condition is multiple sclerosis.
 15. The method of claim 1,wherein the condition is Guillain-Barre Syndrome.
 16. A peptideantagonist of zonulin comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:15, and SEQID NO:23; and wherein said peptide antagonist binds to a zonulaoccludens toxin receptor, yet does not physiologically modulate theopening of mammalian tight junctions.
 17. A method for treatment ofgastrointestinal inflammation comprising administering to a subject inneed of such treatment, a pharmaceutically effective amount of a peptideantagonist of zonulin, wherein said peptide antagonist of zonulincomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:15 and SEQ ID NO:23, wherein saidpeptide antagonist binds to a zonula occludens toxin receptor in theintestine of said subject, yet does not physiologically modulate theopening of mammalian tight junctions in said intestine.